| Sectionl:Gardenia blue staining effect on the anterior capsule of the lens and the safety of the anterior segmentPurpose:To explore the effect of gardenia blue stain on the anterior capsule of the lens and the optimal staining concentration;compare the changes of intraocular pressure,cornea and anterior chamber after staining the anterior capsule with trypan blue.Materials and methods:Eighteen healthy New Zealand white rabbits without eye disease were selected and randomly divided into 6 groups according to different concentrations of gardenia blue stains(5%,4%,3%,2%,1%,0.5%concentration).The anterior capsule of the rabbit lens was stained,and the staining effect was observed.The stained anterior capsule was removed by continuous circular capsulorhexis to determine the optimal dye concentration.Twenty-seven healthy New Zealand white rabbits without eye disease were randomly divided into 3 groups:gardenia blue stain group;bio-blue positive drug group;normal saline group,and the right eye was the experimental eye.The intraocular pressure,anterior chamber inflammation,corneal endothelial injury,and anterior chamber angle pathological changes were measured in 1D,7D,and 14D.The damage of corneal endothelium was detected by alizarin red-trypan blue staining,and the remaining tissues were fixed,embedded,frozen,sectioned,and HE stained,and the pathological changes of the anterior chamber angle were observed with a fluorescence microscope.Results:1.Gardenia blue staining agent can effectively stain the anterior capsule of the lens,and the dyeing concentration is 2%,and the dyeing effect is the best when dyeing for 30s.2.Test the IOP on 1D,7D,and 14D of Gardenia blue stain group,bio-blue positive drug group,and normal saline group,respectively.There was no significant difference in IOP in each group.At the same time,the changes of the anterior chamber of the experimental animals in each group were detected by a slit lamp.The corneal thickness of the experimental animals in each group was uniform,and the depth of the anterior chamber was uniform.No symptoms such as edema,inflammation,and anterior chamber flash were observed.3.Corneal endothelial red-trypan blue staining showed that the corneal endothelium was intact,no edema,no blue staining,no obvious damage to the corneal endothelium,and no obvious inflammation in the anterior chamber angle after HE staining.Conclusion:1.Gardenia blue stain can effectively stain the anterior capsule of the lens,and it will not cause symptoms such as elevated intraocular pressure,corneal edema,and anterior chamber inflammation after anterior chamber injection.2.The stain has no damage to the corneal endothelium,no obvious inflammatory response in the anterior chamber angle and the stain does not cause pathological changes such as inflammation of the anterior chamber angle.Section2:Toxicity Test of Gardenia Blue Stain on Rat RetinaPurpose:After injecting gardenia blue stain into the vitreous cavity,observe whether it has toxic and side effects on the retina.Materials and Methods:Thirty-six healthy SD rats without eye disease were randomly divided into 4 groups:saline group;bio-blue anterior capsule stain group;gardenia blue pigment group;N-methyl-D-aspartic acid In the negative control group,the right eye was used as the experimental eye,and the corresponding reagent was injected into the vitreous cavity of each group of experimental animals.Three experimental animals were sacrificed at 1D,7D,and 14D.Immediately take the material,fix,embed,freeze overnight,prepare frozen sections,collect tissues near the optic nipples for HE staining and TUNEL apoptosis staining,and detect the toxicity of the staining agent on the dead retina side effect.Results:1.Gardenia blue stain group,normal saline group,bio-blue anterior capsule stain group,retinal inner nuclear layer,outer nuclear layer,optic ganglion cell morphology intact,no obvious cell damage,N-methyl-In the D-aspartic acid group,the ganglion cells were intact in shape,the inner core cells were slightly lysed,the inner core layer became thinner,and the outer nuclear layer showed scattered nucleus shrinkage,which caused some damage to the retina.2.Gardenia blue staining group,saline group,bio-blue anterior capsule staining group showed complete cell morphology,no red stained cells,no apoptotic cells in the retinal layer,N-methyl-D-aspartate In the acid experiment group,it can be seen that after experiencing 7D and 14D in the experimental animals,the exosomes are scattered in red colored cells,and the exosomes are scattered in apoptosis.Conclusion:Gardenia blue stain has no obvious damage to retinal cells and no apoptotic cells in the optic nerve layer.It is preliminarily determined that gardenia blue pigment has no toxic and side effects on the retina.Section3:Antioxidant effect of gardenia blue on lens epithelial cellsPurpose:To induce oxidative damage of lens epithelial cells with H2O2,and then study the antioxidant effect of gardenia blue on the injured lens epithelial cells.Material and method:Main instruments and reagents:human lens epithelial cell line SRA01/04,purchased from American ATCC cell bank,Gardenia blue stain(99%)(Shanxi Yikanglong Biotechnology Co.,Ltd.),fetal bovine serum,DMEM High-sugar medium(Germany BI company),pancreatin(U.S.Gibico company),MTT reagent(U.S.Sigma company),electronic balance(Henan Brother Instruments Co.,Ltd.,FA1204B),microplate reader(Biotek Gen5),low-temperature benchtop Centrifuge(Siemens,Germany),inverted phase contrast fluorescence microscope(Nikon,Japan),cell counter,cell counting plate(Wuxi Defan Instruments Co.,Ltd.).Methods:Use H2O2 with a concentration of 100?mol/L,200?mol/L,400?mol/L,800?mol/L on SRA cells for 24 h to determine the optimal concentration of H2O2 for subsequent experiments.In the antioxidant experiment,using gardenia blue solution of 10?mol/L,30?mol/L,50?mol/L,100?mol/L on SRA cells for 1h,H2O2 was added to continue culturing for 24 h to cause oxidative damage to SRA cells by MTT The antioxidant effect of gardenia blue on SRA cells was detected.Results:1.The viability of SRA cells decreased with the increase of H2O2 concentration,showing a dose-dependent.The H2O2 concentration of subsequent experiments was selected to be 200?mol/L.2.The results of MTT assay showed that after oxidative damage to SRA cells in the H2O2 group,the cell survival rate(49.85+6.49)%was significantly reduced,compared with the normal control group(99.55+3.78)%,the difference was statistically significant(P<0.001).After pretreatment with Gardenia Blue solution for 1h,the survival rate of SRA cells treated with H2O2 was improved,the cell survival rate of 10?g/mL Gardenia Blue+H2O2 group(55.80±2.87)%;30?g/mL Gardenia Blue+H2O2 group The cell survival rate was(60.11±2.43)%;the cell survival rate in the 50?g/mL Gardenia Blue+H2O2 group(64.28±2.26)%;the cell survival rate in the 100?g/mL Gardenia Blue+H2O2 group(61.97±2.61)%.Compared with the H2O2 model group,the differences were statistically significant(bothP?0.05)?Conclusion:1.The viability of SRA cells decreased with the increase of H2O2 concentration,showing a dose-dependent.The H2O2 concentration of subsequent experiments was selected to be 200?mol/L.2.Gardenia blue solution has antioxidant effect on SRA cells. |
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